Since the last blog post, I have made progress in my scientific research. Before taking on new experiments, my research mentor and I decided to sort through old data I had previously collected in an aim to figure out if any experiments, I had conducted in the past, needed to be repeated. This was done to make sure that I had enough replicates of my experimental data to produce more statistically significant results. After reviewing my data, I chose to replicate an RNAi experiment using my model organism of choice N2, C.elegans hermaphrodites. Again, N2 C. elegans are a wild-type strain. What this experiment involved was using a positive control (npp-19), a negative control (L440), and my gene of interest (M05D6.2) to view the amount of progeny that was produced after five days of experimentation.
To set up this experiment, the first thing I did was grow my bacterial cultures, which contained siRNA, that would be used to silence a targeted gene, on a Monday evening. The bacteria were then incubated for 16 hours. After 16 hours a 100 microliter spot of each bacterium was placed on a RNAi plate and incubated at room temperature for 24 hours. On Wednesday morning I set up my experiment, placing 5 L4 N2 C.elegans hermaphrodites on each plate and incubating them at 22 degrees Celsius overnight. On Thursday I picked off the matured C. elegans hermaphrodites, leaving on the plates the eggs which had been laid. Then on Friday I counted the amount of hatched and unhatched eggs on each plate. The results of this experiment showed that silencing the M05D6.2 gene in N2 hermaphrodite C.elegans had no effect in progeny count. This is exactly the result we wanted to see. Showing that this gene does not play a role in hermaphrodite fertility helps solidify our previous conclusion that the M05D6.2 gene plays a role in male fertility.
Along with collecting the data from this experiment, I have also been performing statistical analysis using a program called R. The next step in my research will be to learn how to use a technique known as microscopy to view internally the impact of the RNAi treatments on male C. elegans as well as hermaphrodites. I will also be setting up a new cross between two different C. elegans strains to see how the fertility of these two strains are effected after being exposed to the RNAi treatment.